Background: Cosmos bipinnatus is an important medicinal plant with antioxidative, antigenotoxic, anti-infl ammatory, and antiproliferative eff ects on several cancer cell lines. It has great potential for development as a promising cancer chemo-preventive agent. Hairy ROOT-based culture technology is a new sustainable production platform for producing specifi c pharmaceutical secondary metabolites. Objectives: The current study developed and introduced a reliable transformation system for C. bipinnatus by optimization of aspects important in transformation frequency using Agrobacterium rhizogenes. Methods: Five bacterial strains, including ATCC 15834, ATCC 31798, A7, MAFF-02-10266, and MSU440, 2 explant types (leaf and stem), and 2 co-cultivation media (full MS and ½ MS) were examined. Genomic DNA was extracted using a modifi ed CTAB protocol from putative transgenic ROOT lines and the control ROOT. Transgenic hairy ROOT lines were approved by means of Polymerase Chain Reaction (PCR) using specifi c rolB gene primers. Results: The highest ratio of genetically TRANSFORMED ROOT induction was found from leaf explants using A. rhisogenes strains ATCC15834 and MSU440 (72% to 73%). When ½ MS medium was used as a co-cultivation medium, a signifi cant increase in transformation frequency (84%) was observed. Conclusions: The MSU440 Agrobacterium strain and ½ MS co-cultivation medium could signifi cantly improve genetic transformation effi ciency for establishment of hairy ROOT-based cultures for C. bipinnatus.